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Insights into product release in the hydrolysis reaction catalyzed by
the bacterial deubiquitinase SdeA
Sebastian Kenny^1 , Kwame Brown^1 , Chittaranjan Das^1 (^1) Department of Chemistry, Purdue University, WL, IN, 47905, USA Crystal structure of the Cys-Ala mutant of the deubiquitinase (DUB) domain of the Legionella pneumophila effector SdeA (SdeADUB) reveals intermolecular contacts of the prokaryotic DUB with the ubiquitin (Ub) product. Most of the interactions are preserved in the product bound structure relative to the complex of the DUB with the suicide inhibitor ubiquitin vinylmethyl ester (Ub-VMe), a complex whose structure closely mimics the structure of the substrate bound state of SdeADUB. These interactions are also preserved in the product bound state of the enzyme in solution as revealed by NMR titration studies. Isothermal titration calorimetry (ITC) and NMR titration data reveal a significant difference in affinity for Ub between the wild type and the Cys-Ala mutant enzyme, with the mutant displaying a significantly higher affinity. We attribute this difference to repulsive interactions between the thiolate ion of the catalytic Cys and the carboxylate ion of the terminal Gly76 residue of the Ub, a product feature that results upon hydrolysis of the isopeptide bond linking Ub to a protein a target. Thus, product release is likely facilitated by a repulsive interaction between the catalytic Cys (thiolate anion) and the carboxylate group produced from the hydrolysis of the isopeptide bond of the ubiquitinated substrate. Similar repulsive interactions may underlie a general mechanism of product release in hydrolysis of deubiquitinases and hydrolases of ubiquitin like protein modifiers where extensive protein-protein interactions are utilized in the enzyme-substrate engagement.
In Search of the Syk Phosphorylation Mechanism
Jacob Jerald Kinnun^1 , Chao Feng^1 , Duy Hua^1 , and Carol Beth Post^1 (^2) Department of Medicinal Chemistry and Molecular Biology, Purdue University, WL, IN, 47905, USA Spleen tyrosine kinase (Syk) has a central role in the transmission of activating signals within B-cells of the adaptive immune system. Abnormal function of Syk is implicated in many hematopoietic tumors and autoimmune diseases. Therefore, the activation mechanism of Syk is of particular biological and pharmacological interest. Our Syk fragment comprises two SH2 domains separated by a linker region. Nuclear magnetic resonance spectroscopy (NMR) and molecular dynamics simulations (MD) have revealed that there is an increased disorder in SH2-SH2 domain structure, or uncoupling of the two SH domains, when Syk is phosphorylated at the linger region. This behavior is the basis of a novel phosphorylation mechanism that is entropically driven. The question remains how phosphorylation in the linker region induces domain separation. MD results suggest, upon phosphorylation, the linker region has increased interaction with a few basic residues (R67 & K164) and R45 changes interaction partners. To test if these interactions are important we substituted these residues with alanine (a small uncharged amino acid). If these interactions are important; mutations should affect the uncoupling mechanism with phosphorylation mimics of Syk. However, we still observed domain uncoupling in NMR measurements. One potential way to prevent interaction after phosphorylation is to increase salt concentration, which screens the charge introduced in phosphorylated residues. The results of introducing salt to uncovering the phosphorylation mechanism of Syk will be presented and future directions discussed.
High-throughput protein correlation profiling reveals evolutionary
diversification of protein complexes in oligomeric states across
plant species
Youngwoo Lee^1 , Uma K. Aryal^2 , Zachary A. McBride^3 , Thomas W. Okita^3 , and Daniel B. Szymanski^1 (^1) Department of Botany and Plant Pathology, Purdue University, WL, IN, 47905,USA (^2) Purdue Proteomics Facility, Purdue University, WL, IN, 47905, USA (^3) Department of Biological Sciences, Purdue University, WL, IN, 47905,USA (^3) Institute of Biological Chemistry, Washington State University, Pullman, WA, 99164,USA Multiprotein complexes are essential for coordinating cellular processes evolved through millions of years. However, the components, conservation and functions of these protein complexes remain enigmatic in planta. To analysize the preservation of protein complexes across plant species, we employed a new combination method of protein correlation profiling (PCP) and shutgon proteomics that provides large-scale datasets on protein oligomerization. This technology permited a high-throughtput comparative analysis of the elution profiles of mass spectrometry-characterized proteins among diverse plant models. To test for species-specific differences and similarities in ubiquity and dimensions of protein complexes across plant species, here four plant species were selected both for their distance in evolutionary divergence and also for their relevance as model organisms to biology research and crop production: two Malvids (leaves of Arabidopsis thaliana and fibers of Gossypium hirsutum), a Fabid (leaves of Glycine max) among dicot plants, and one monocot species of the Poales (aleurone layer of Oryza sativa). Protein complexes in the four representative species were fractionized by size exclusion chromatography (SEC) and followed by a label-free quantitative mass spectrometry analysis. For cross-species comparisons, annotated proteins in each plant species were combined into orthologous groups using Phytozome V12 plant ortholog information, and then assessed by the analysis of variance (ANOVA) F-test. Interestingly, some of orthologous protein complexes are not conserved in terms of complex size, suggesting evolutionary divergence may have shaped protein number or size distributions. To get more insight into this protein complex evolution, protein complex composition has been accomplished in Arabidopsis and rice, and then the degree of overlap of protein subunit composition will be analyzed.
Identifying critical genes and microRNAs that potentiate
KRAS ;p53-driven lung tumorigenesis
Chennan Li^1 , Abigail K. Mesfin^1 , Nadia A. Lanman^1 , and Andrea L. Kasinski 1, (^1) Department of Biological Sciences, Purdue University, WL, IN, 47905,USA (^2) Purdue Center for Cancer Research, Purdue University, WL, IN, 47905, USA Lung cancer is the leading cause of cancer-related deaths. KRAS and TP53 are two of the most commonly mutated genes in lung cancer, and their mutations are well recognized drivers of tumorigenesis. Directly targeting these drivers still remains a challenge for cancer therapeutics. Instead, targeting genes that potentiate KRAS/p53-driven tumorigenesis is likely an effective alternative. Therefore, we hypothesize that loss of certain genes and microRNAs can drive transformation of two non-tumorigenic KRAS;p53/Kras-mutated mammalian lung systems into cancerous states. To address this, we performed CRISPR knockout screens in these two systems using the small guide RNA (sgRNA) libraries. We identified several sgRNAs that could support anchorage-independent growth of the originally anchorage-dependent human bronchial epithelial cells that harbored KRASG12V;sh-p53 (HBEC-KP), and in concordance with this, these sgRNAs were enriched in two-dimensional culture over four-months of growth. KrasG12D;Cas9 mouse line was also generated and genome-wide knockouts will be induced directly in murine lungs for evaluating genetic contributions to tumor development.
Engineering a Color Palette of FRET-BRET ATP Sensors
Sehong Min^1 , Mathew Tantama^1 (^1) Department of Chemistry, Purdue University, WL, IN, 47905, USA ATP, a key player in energy metabolism, is involved in various cellular processes and is shown to be an indicator of cellular health. There are several methods to measure ATP levels, and genetically-encoded sensors are excellent tools for visualizing energy metabolism in live cells. Currently, however, there has been a lack of color options. By building new variants of hybrid FRET-BRET (Förster or bioluminescence resonance energy transfer) ATP sensors, our goal is to increase the flexibility and utility of these tools with different experimental formats. For example, differently colored sensors could be used to simultaneously monitor ATP levels in different individual cells or cell types as well as in different compartments within a single cell. Here, we have developed ATP sensors which employ different fluorescent protein FRET pairs in conjunction with the NanoLuc luciferase. We characterized the both the fluorescence and bioluminescence purified sensor proteins in response to ATP. Furthermore, we carried out live-cell imaging experiments to validate that our sensors can be used for ratiometric quantification of ATP levels. These new color variants of hybrid FRET-BRET ATP sensors will be an effective method for studying energy metabolism both in vitro and in vivo.
Cryo-EM Structural Analysis of Human Deubiquitinating Enzyme
USP7 Illustrates Tethered-Rheostat Mechanism of Catalysis
Corey Moore^1 , Brenda Gonzalez^1 , Frank Vago^1 , Kunpeng Li^1 , Wen Jiang^1 , Andrew Mesecar1, (^1) Department of Biological Sciences, Purdue University, WL, IN, 47905, USA (^2) Department of Biochemistry, Purdue University, WL, IN, 47905, USA Ubiquitin-specific protease 7 (USP7) is a human deubiquitinating enzyme that is implicated in numerous cancers and neurodegenerative diseases. The family of USPs all contain a fingers-palm-thumb catalytic domain and a Cys-His-Asp catalytic triad. Due to their similarity in sequence and structure, structure-activity relationship studies for therapeutic design has been a challenge. USP7 is a unique member of this family of enzymes in that it has 7 domains, many of which are not shared by closely related enzymes. These domains are hypothesized to work in concert, exhibiting a large degree of conformational heterogeneity and unique intramolecular regulation. Due to these features, the field has enjoyed limited success in x-ray crystallographic studies, relying on individual domain truncations. This has left researchers to piece together the mechanism of catalysis through biochemical analysis alone. The following work represents the first comprehensive structural data to support USP7’s hypothesized tethered-rheostat mechanism of catalysis. Further, the structural analysis herein describes the location of the TRAF and HUBL1-5 domains in relation to the catalytic domain, which has eluded researchers for the past decade.
Time-resolved crystallographic measurements elucidating the
mechanism of bacterial HMG-CoA reductase
Vatsal Purohit^2 , Calvin Steussy^2 , Tim Schmidt^2 , Chandra J. Critchelow^2 , Cynthia Stauffacher^2 , Tony Rosales^2 , Paul Helquist^2 , Olaf Weist^2 (^1) Department of Biological Sciences, Purdue University, WL, IN, 47905, USA (^2) Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, 46556, USA HMG-CoA reductase (HMGR) is the rate limiting enzyme in the Mevalonate pathway that converts 3-hydroxy-3-methylglutarylCoA (HMG-CoA) into Mevalonate, a precursor compound used in isoprenoid biosynthesis. The enzyme is the target for cholesterol-lowering drugs and a potential target for novel antibacterials in gram-positive bacteria. Previous studies have suggested that its substrate HMG-CoA undergoes conversion into two intermediates, Mevaldyl-CoA and Mevaldehyde, before it is converted into Mevalonate. However, only Mevaldyl-CoA, has been structurally observed. Current work on HMGR focuses on identifying conditions that are suitable for running the enzymatic reaction in the crystal and also altering the rate of the reaction within the HMGR crystal to trap and observe the reaction intermediates formed during catalysis thereby testing the reaction mechanism model. By doing so, we also hope to identify the structural and chemical changes that facilitate the enzymatic reaction in HMGR.
Dimerization of the yeast Isoprenylcysteine Carboxyl
Methyltransferase, Ste
Anna Ratliff^1 , Sahej Bains^1 , Srinivas Chakravarthy^2 ,Amy Funk^1 , Amy Griggs^1 , and Christine A. Hrycyna1, (^1) Department of Chemistry, Purdue University, WL, IN, 47905, USA (^2) Argonne National Laboratory, Lemont, IL, 60439, USA (^3) Purdue Center for Cancer Research, Purdue University, WL, IN, 47905, USA Isoprenylcysteine carboxyl methyltransferase (Icmt) is an membrane protein localized to the ER that is responsible for the carboxyl methylesterification of CaaX proteins. Currently little is known about the structure and mechanism of Icmt. Our model, the yeast Icmt Ste14, is comprised of six transmembrane domains in which TM1 contains a dimerization motif, G31XXXG35XXXG39. Previous studies proposed Ste14 forms functional higher order oligomers. Thus, to determine the sequence and structural determinants for dimerization, we used cysteine-scanning mutagenesis to generate single cysteine mutants for every residue in TM1. Each mutant was characterized via immunoblot analyses to assess expression, activity, and the ability to form dimers. Additionally, several of the residues proposed to lie on the same face of TM1 formed dimers upon the addition of sulfhydryl specific cross-linkers. We further validated the homodimerization of wildtype Ste14 with SEC-MALS and SAXS. Together, we suggest that functional form of the Ste14 Icmt is, in fact, a homodimer and will ultimately be useful as we further explore the mechanism of action of Ste14.
Structural Insights into Phospholipase Cε Function
Ngango Yvon Rugema1,2, Sean P. Pearson^1 , William Mbongo^3 , Hannah O'Neill^1 , Keshav Prahalad^1 , Liz Garland-Kuntz^1 , Monita Sieng^1 , & Angeline M. Lyon1,2, (^1) Department of Chemistry, Purdue University, WL, IN, 47905, USA (^2) Interdisciplinary Life Sciences Program, Purdue University, WL, IN, 47905, USA (^3) Department of Biological Sciences, Purdue University, WL, IN, 47905, USA Phospholipase Cε (PLCε) is a member of the PLC family of enzymes that hydrolyze phosphatidylinositol lipids downstream of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). PLCε is unique among the PLC superfamily as it contains an N-terminal CDC25 domain, which has guanine nucleotide exchange factor (GEF) activity towards small G proteins Rap1A, and two C-terminal Ras association (RA) domains. The best characterized pathway leading to PLCε activation is mediated through β-adrenergic receptor (βARs) signaling. Stimulation of these receptors culminates in the activation of the small GTPase, Rap1A, which binds directly to PLCε and translocates the complex to the Golgi. There, PLCε hydrolyzes phosphatidylinol-4-phosphate (PI4P). Prolonged activation of this pathway results in increased expression of hypertrophy-related genes. However, how the structural basis of PLCε activity under basal conditions and upon activation by Rap1A are unknown. Towards this end, we have obtained the first high-resolution insights into a catalytically active PLCε variant. These studies, together with biochemical analysis of the intramolecular contacts observed in the structure, provide the first molecular insights into this enzyme.
Molecular Mechanism of Rap1A-Dependent Activation of
PLCepsilon
Monita Sieng^1 , Elisabeth E. Garland-Kuntz^1 , Arielle Selvia^1 , Andrea Marti^1 and Angeline M. Lyon1, (^1) Department of Chemistry, Purdue University, WL, IN, 47905, USA (^2) Department of Biological Sciences, Purdue University, WL, IN, 47905, USA Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce diacylglycerol (DAG) and inositol phosphates, leading to the activation of protein kinase C (PKC) and downstream signaling pathways, including cell growth and survival. The PLCepsilon subfamily is a key player in cardiovascular function, where it contributes to maximum contractility. However, prolonged activation of PLCepsilon results in cardiac hypertrophy and heart failure through its ability to regulate the expression of hypertrophic genes. This process is regulated by the small GTPase Rap1A, which is activated downstream of Beta-adrenergic receptors. Rap1A binds to the C-terminal Ras association (RA) domain of PLCepsilon, simultaneously translocating the complex to the perinuclear region and activating PLCepsilon. PLCepsilon also contains an N-terminal CDC domain, which has guanine nucleotide exchange factor (GEF) activity for Rap1A resulting in a feed forward activation loop and sustained lipid hydrolysis. However, the molecular mechanism of this process is not known. In this work, we seek to characterize the interactions between Rap1A and PLCepsilon using structural and functional studies to map the Rap1A binding site on PLCepsilon and determine whether activation results in conformational changes that release autoinhibition and/or increase membrane association. These studies provide the first molecular details of the Rap1A-dependent activation of PLCepsilon and open the door to the development of new therapeutic strategies for treating cardiac hypertrophy.
2.6 Å Structure of Tulane Virus With Minor Mutations Leading to
Receptor Change
Chen Sun^1 , Li Kunpeng^1 , Frank S. Vago^1 , Wen Jiang^1 , Pengwei Huang, Ming Tang, Xi Jiang(Cincinnati Children’s Hospital Medical Center, Division of Infectious Diseases) (^1) Department of Biological Sciences, Purdue University, WL, IN, 47905, USA (^1) Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229, USA It has been a common practice to eliminate mutations during virus propagation in vitro. However, for low yield viruses, like Tulane virus (TV) and Hepatitis B virus (HBV), numerous efforts have been made to increase the virus titer at every steps of virus propagation and purification. In this study, we were able to obtain a high yield TV mutant and whole genome sequencing has revealed several mutation sites. A 2.6 Å resolution structure of the TV mutant was solved using cryo-EM with EM density consistent with the sequence variations. Most importantly, via the enzyme-linked immunosorbent assay (ELISA) experiment, the TV mutant has been found to lost the binding ability to its original cellular receptor, the histo-blood group antigens (HBGAs). Therefore, it is appealing to propose that it may have switched to a new receptor to better adapt the cell culture system.
Molecular determinants of the differential modulation of Cav1.2 and
Cav1.3 by nifedipine and FPL 64176
Yuchen Wang^1 , Shiqi Tang^1 , Kyle E. Harvey^1 , Amy E. Salyer^1 , T. August Li^1 , Emily K Rantz^1 , Markus A. Lill^1 , and Gregory H. Hockerman^1 (^1) Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, WL, IN, 47905, USA Nifedipine and FPL 64176 (FPL), which block and potentiate L-type voltage-gated Ca2+ channels respectively, more potently modulate Cav1.2 than Cav1.3. To identify potential strategies for developing subtype-selective inhibitors, we investigated the role of divergent amino acid residues in transmembrane domains IIIS5 and the extracellular IIIS5-3P loop region in modulation of these channels by nifedipine and FPL. Insertion of the extracellular IIIS5-3P loop from Cav1.2 into Cav1.3 (Cav1.3+) reduced the IC50 of nifedipine from 289 nM to 101 nM, and substitution of S1100 with an A residue, as in Cav1.2, accounted for this difference. Substituting M1030 in IIIS5 to V in Cav1.3+ (Cav1.3+V) further reduced the IC50 of nifedipine to 42 nM. FPL increased current amplitude with an EC50 of 854 nM in Cav1.3, 103 nM in Cav1.2, and 99 nM in Cav1.3+V. In contrast to nifedipine block, substitution of M1030 to V in Cav1.3 had no effect on potency of FPL potentiation of current amplitude, but significantly slowed deactivation in the presence and absence of 10 μM FPL. FPL had no effect on deactivation of Cav1.3/DHPi, a channel with very low sensitivity to nifedipine block (IC50 ~ 93 μM), but did shift the voltage-dependence of activation by ~-10 mV. We conclude that the M/V variation in IIIS5 and the S/A variation in the IIIS5-3P loop of Cav1.2 and Cav1.3 largely determine the difference in nifedipine potency between these two channels, but the difference in FPL potency is determined solely by divergent amino acids in the IIIS5-3P loop.
Enhancement for MAINMAST, De Novo Main-Chain Tracing Method:
Symmetric Multi-Chain Modeling, Local Refinement, and Graphical
User Interface
Genki Terashi^1 , Yuhong Zha^2 , and Daisuke Kihara1, (^1) Department of Biological Sciences, Purdue University, WL, IN, 47905, USA (^2) School of Computer Science, Carnegie Mellon University, Pittsburg, PA, 15213, USA (^3) Department of Computer Science, Purdue University, WL, IN, 47905, USA The significant progress of the cryo-EM poses a pressing need for software for structural interpretation of EM maps. Particularly, protein structure modeling tools are needed for EM maps determined around 4 Å resolution, where finding main-chain structure and assigning the amino acid sequence into EM map are still challenging problems. We have developed a de novo modeling tool named MAINMAST (MAINchain Model trAcing from Spanning Tree) for EM maps for this resolution range. MAINMAST builds main-chain traces of a protein in an EM map from a tree structure constructed by connecting points with a high density in the map without referring to known protein structures or fragments. The method has substantial advantages over the existing methods: i) MAINMAST directly constructs protein structure models from an EM density map without requiring reference structures; ii) The procedure is fully automated and no manual setting is required; iii) a pool of models are produced, from which a confidence score is computed that indicates accuracy of structure regions. Here, we report substantial improvements of MAINMAST in three aspects. The largest improvement is that the method now can perform automatic map segmentation and structure modeling for symmetrical multi-chain complexes. The tree-graph structure that connects dense points are traced for multiple chains simultaneously in a symmetric fashion. Figure 1 and 2 are showing the examples of the multi-chain segmentation by MAINMAST and Segger v1.9.5 (plugin in UCSF Chimera molecular visualization software) on EMD-6551 (Magnesium channel CorA at 3.8Å resolution) and EMD-8118 (TRPV1 at 3.28Å resolution). In these two maps, MAINMAST successfully find the individual protein regions from the EM maps. Moreover, the accuracy of a model is significantly improved by a new implementation of local sequence matching and structure refinement. The local matching protocol is also useful for identifying missing regions in a structure model, i.e. regions with a low density, in an EM map. Finally, we developed a software plugin of MAINMAST for the UCSF Chimera, so that users can monitor structures at each step of a modeling procedure. The major functionalities include to generate and to display tree structures from local dense points in the map, main-chain traces, and reconstructed all-atom models. Through the interface, users can easily control parameters of MAINMAST and save and restore sessions.
Conformational Dynamics Contribute to Phospholipase Cbeta
Activity
Michelle Van Camp^1 , Elisabeth Garland-Kuntz^1 , Angeline M. Lyon1, (^1) Department of Biological Sciences, Purdue University, WL, IN, 47905, USA (^2) Department of Biological Sciences, Purdue University, WL, IN, 47905, USA Phospholipase C (PLC) enzymes hydrolyze the lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the second messengers inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Production of these second messengers leads to many diverse physiological responses, including vascular smooth muscle contraction and inflammation. Previous structural and functional studies of PLCbeta have revealed a highly conserved catalytic core that adopts a compact, globular structure and forms the minimal fragment that retains lipase activity. However, a growing body of evidence suggests that the PLCbeta PH domain, which interacts with EF hands and TIM barrel of the catalytic core in crystal structures, may be flexible in solution. Using a combination of small angle x-ray scattering (SAXS), cross-linking studies, and biochemical assays, we provide the first structural data demonstrating that the PH is in fact conformationally flexible in solution. The PH domain adopts two major states: an open state wherein the PH domain is extended away from the core, and a closed state consistent with the crystal structures. We also provide evidence that these conformational states are functionally significant. These findings provide new evidence of conformational heterogeneity of PLC enzymes in solution and reveal new insights into their roles in cell signaling and cardiovascular disease.
